Retinal degeneration is a leading cause of vision impairment and blindness worldwide and medical care for advanced disease does not exist. Stem cell-derived retinal organoids (RtOgs) became an emerging tool for tissue replacement therapy. However, existing RtOg production methods are highly heterogeneous. Controlled and predictable methodology and tools are needed to standardize RtOg production and maintenance. In this study, we designed a shear stress-free micro-millifluidic bioreactor. We used a stereolithography (SLA) 3D printer to fabricate a mold from which Polydimethylsiloxane (PDMS) was cast. The multi-chamber bioreactor design and fabrications methods easily combined micro and millimeter features with very low cost and short manufacturing time. We optimized the chip design using in silico simulations and in vitro evaluation to optimize mass transfer efficiency and concentration uniformity in each culture chamber. We successfully cultured RtOgs on an optimized bioreactor chip for 37 days. We also characterized the RtOgs produced by static dish culture and chip culture methods using qualitative and quantitative techniques. Phase contrast imaging showed that both conventional and chip-cultured RtOgs developed a transparent outermost surface structure. Fluorescence lifetime imaging (FLIM) showed that RtOgs on the chip had significantly lower long lifetime species (LLS) ratio than static cultured ones, which demonstrated that bioreactor cultured RtOgs exhibited less oxidative stress. RtOgs in bioreactor culture demonstrated higher NADH signal overall, but both bioreactor and conventional cultures showed similar free/bound NADH ratio over time, which indicated normal differentiation time course. RtOg gene expression was examined by fluorescence imaging and quantitative polymerase chain reaction (qPCR) analyses. RtOgs in both groups showed thick nuclear outer layers expressing CRX on day 120 of differentiation. The gene profiling showed both groups expressed retinal progenitor genes and most of the tested photoreceptor markers. We, therefore, validated an autonomous micro-millifluidic device with significantly reduced shear stress and lower oxidative stress to produce RtOgs of equal or greater quality than those maintained in conventional static culture.
Abstract Retinal progenitor sheet transplants have been shown to extend neuronal processes into a degenerating host retina and to restore visual responses in the brain. The aim of this study was to identify cells involved in transplant signals to retinal degenerate hosts using computational molecular phenotyping (CMP). S334 ter line 3 rats received fetal retinal sheet transplants at the age of 24–40 days. Donor tissues were incubated with slow‐releasing microspheres containing brain‐derived neurotrophic factor or glial cell‐derived neurotrophic factor. Up to 265 days after surgery, eyes of selected rats were vibratome‐sectioned through the transplant area (some slices stained for donor marker human placental alkaline phosphatase), dehydrated and embedded in Eponate, sectioned into serial ultrathin datasets and probed for rhodopsin, cone opsin, CRALBP (cellular retinaldehyde binding protein), l ‐glutamate, l ‐glutamine, glutathione, glycine, taurine, γ‐aminobutyric acid (GABA) and DAPI (4′,6‐diamidino‐2‐phenylindole). In large transplant areas, photoreceptor outer segments in contact with host retinal pigment epithelium revealed rod and cone opsin immunoreactivity whereas no such staining was found in the degenerate host retina. Transplant photoreceptor layers contained high taurine levels. Glutamate levels in the transplants were higher than in the host retina whereas GABA levels were similar. The transplant inner nuclear layer showed some loss of neurons, but amacrine cells and horizontal cells were not reduced. In many areas, glial hypertrophy between the host and transplant was absent and host and transplant neuropil appeared to intermingle. CMP data indicate that horizontal cells and both glycinergic and GABAergic amacrine cells are involved in a novel circuit between transplant and host, generating alternative signal pathways between transplant and degenerating host retina.
Abstract The aim of this study was to establish synapses between a transplant and a degenerated retina. To tackle this difficult task, a little‐known but well‐established CNS method was chosen: trans‐synaptic pseudorabies virus (PRV) tracing. Sheets of E19 rat retina with or without retinal pigment epithelium (RPE) were transplanted to the subretinal space in 33 Royal College of Surgeons (RCS) and transgenic s334ter‐5 rats with retinal degeneration. Several months later, PRV‐BaBlu (expressing E. coli β‐galactosidase) or PRV‐Bartha was injected into an area of the exposed superior colliculus (SC), topographically corresponding to the transplant placement in the retina. Twenty normal rats served as controls. After survival times of 1–5 days, retinas were examined for virus by X‐gal histochemistry, immunohistochemistry and electron microscopy. In normal controls, virus was first seen in retinal ganglion cells and Müller glia after 1–1.5 days, and had spread to all retinal layers after 2–3 days. Virus‐labeled cells were found in 16 of 19 transplants where the virus injection had retrogradely labeled the topographically correct transplant area of the host retina. Electron microscopically, enveloped and nonenveloped virus could clearly be detected in infected cells. Enveloped virus was found only in neurons. Infected glial cells contained only nonenveloped virus. Neurons in retinal transplants are labeled after PRV injection into the host brain, indicating synaptic connectivity between transplants and degenerated host retinas. This study provides evidence that PRV spreads in the retina as in other parts of the CNS and is useful to outline transplant–host circuitry.
The ultrastructure of rat to rat and human to rat long-term transplants of retinal cell transplants has been investigated. The human transplants were examined at 30 to 41 weeks of total age after conception. Rat homotransplants were nine to ten weeks of age after conception in four cases and 20 weeks in one case. Xenotransplanted rats were immunosuppressed with Cyclosporin A.Both xeno-and homotransplants can develop in the epiretinal or the subretinal space. The development is often heterogeneous. Photoreceptor cells can form both inner and outer segments as well as synaptic terminals. In regions corresponding to the inner plexiform layer, the adult complement of synapses has been seen, including advanced features like serial synapses as well as reciprocal synapses at bipolar cell dyads. Incompletely differentiated synapses of both amacrine and bipolar cell types have been observed, especially in rat epiretinal transplants. Ganglion cell processes have not been identified with certainty. Transplants from both species develop according to their intrinsic, genetically determined time table. Rat subretinal transplants tend to become more developed than epiretinal grafts, but only epiretinal grafts have so far been seen to make contacts with the host retina.Connections between the transplant and the host have been seen in a number of cases, but are not a regular feature. Usually, there are no obvious specializations where transplant cells touch host photoreceptor cells or pigment epithelium. Graft cells have occasionally been found within the host retina, and nerve cell processes have been observed to cross the membrane separating epiretinal transplants and host.Human retinal cells from first trimester embryos and rat El5 retinal cells in long-term xenotransplants are thus able to survive, differentiate, and develop synapses of normal appearance as well as complex neuronal circuits.
4. Immunohistochemistry for Molecular Phenotyping 1. Transplantation S334ter-line-3 rats (age 0.8 1.4 months) with fast retinal degeneration received E19 fetal retinal sheet transplants derived from transgenic rats expressing hPAP (human placental alkaline phosphatase). Some donor retinas were coated with BDNF microspheres or GDNF microspheres before implantation (Seiler et al. 2008b). Rat eyes were imaged by 3D-Ocular Coherence Tomography (OCT) 0.4 2.2 months after surgery to determine transplant placement and layering (Seiler et al. 2010b). Thirteen transplanted rats were selected and sacrificed at 2.3 – 8.5 months after transplantation (age 3.4 – 9.9 months). Eight of these rats were recorded by electrophysiology for visual responses in the superior colliculus (below).
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